The present invention concerns cloned human adenosine receptors of the A1, A2 and A3 class and their subtypes. The cloned dog A1 and A2a adenosine receptors have been reported. See F. Libert, et al., (1989) Science 244:569-572, C. Maennant, et al., Biochem. Biophys. Res. Comm., (1990) 173:1169-1178, and F. Libert, et al. (1991) EMBO J. 10:1677-1682. The cloned rat A1 adenosine receptor has been reported by L. C. Mahan, et al., (1991) Mol. Pharm. 40:1-7 and S. M. Reppert, et al., (1991) Mol. Endocrin. 5:1037-1048. We have now found that the human A1 adenosine receptor differs by 18 amino acids from the dog A1 sequence and 16 amino acids from the rat A1 sequence. The human A2a adenosine receptor differs by 28 amino acids from the dog A2a sequence.
Adenosine is a naturally occurring nucleoside which exhibits diverse and potent physiological actions in the cardiovascular, nervous, pulmonary, renal and immune systems. Adenosine has been demonstrated to terminate superventricular tachycardia through blockage of atrioventricular nodal conduction (J. P. DiMarco, et al., (1985) J. Am. Col. Cardiol. 6:417-425, A. Munoz, et al., (1984) Eur. Heart J. 5:735-738). Adenosine is a potent vasodilator except in the kidney and placenta (R. A. Olsson, (1981) Ann. Rev. Physiol. 43:385-395). Adenosine has been implicated as a preventative agent and in treatment of ventricular dysfunction following episodes of regional or global ischemia (M. B. Forman and C. E. Velasco (1991) Cardiovasc. Drugs and Therapy 5:901-908) and in cerebral ischemia(M. C. Evans, et al., (1987) Neurosci. Lett. 83:287, D. K. J. E., Von Lubitz, et al., (1988) Stroke 19:1133).
The instant invention also concerns an assay protocol which can be used for identifying and evaluating substances that bind to human adenosine receptors. The assay can be utilized to identify adenosine receptor agonists and antagonists and determine their binding affinity (R. F. Bruns, et al., (1983) Proc. Natl. Acad. Sci. USA 80:2077-2080; R. F. Bruns, et a1.,(1986) Mol. Pharmacol. 29:331-346; M. F. Jarvis, et al. (1989) J. Pharma. Exp. Therap. 251:888-893; K. A. Jacobson et al., (1989) J. Med. Chem. 32:1043-1051). Such adenosine receptor agonists, antagonists and binding enhancers have been identified and implicated for usage in the treatment of physiological complications resulting from cardiovascular, renal and neurological disorders. Adenosine receptor agonists have been identified for use as vasodilators ((1989) FASEB. J. 3(4) Abs 4770 and 4773, (19910 J. Med. Chem. (1988) 34:2570), antihypertensive agents (D. G. Taylor et al., FASEB J. (1988) 2:1799),and anti-psychotic agents (T. G. Heffner et al., (1989) Psychopharmacology 98:31-38). Adenosine receptor agonists have been identified for use in improving renal function (R. D. Murray and P. C. Churchill,(1985) J. Pharmacol. Exp. Therap. 232:189-193) Adenosine receptor allosteric or binding enhancers have shown utility in the treatment of ischemia, seizures or hypoxia of the brain (R. F. Bruns, et al. (1990) Mol. Pharmacol. 38:939-949; C. A. Janusz, et al., (1991) Brain Research 567:181-187). The cardioprotective agent, 5-amino-4imidazole carboxamide (AICA) ribose has utility in the treatment of ischemic heart conditions, including unstable angina and acute s myocardial infarction (H. E. Gruber, et al. (1989) Circulation 80: 1400-1414). Previous methods to date have proven inferior due to the presence of multiple subtypes present in the animal tissue utilized (R. F. Bruns et al., (1986) Mol. Pharm. 29:33 1-346) and the differences between species in the affinity for adenosine analogs and the physiological effects of adenosine (Ukera, et al., (1986) FEBS Lett, 209:122-128). Pure adenosine receptors make possible the identification and evaluation of compounds which have unique affinity for a single receptor subtype. Moreover, because of the variable effects of adenosine documented in other species, the utilization of human adenosine receptor subtypes is advantageous for the development of human therapeutic adenosine receptor agonists, antagonists or enhancers.